1.1 Field of The Invention
The invention pertains to methods and devices for detecting targeted microorganisms such as bacteria by inducing bioluminescence in bioreporter cells. Genetically engineered bacteriophage are employed to infect target bacteria in the presence of genetically engineered bioreporter cells. The bioreporter cells respond by producing light upon stimulation by an inducer. The inducer is produced as a result of infection of the target bacteria by the bacteriophage.
1.2 Description of the Related Art
Current technology focusing on the development of biologically-based detection systems has prompted efforts to address the need for methods for detecting specific microbial pathogens. Numerous methods for determining the presence of microbial contaminants have been used over the years; typically, culture methods were employed in the past but these methods were slow and inefficient. Recent developments in bioreporter technology have prompted use of genetically engineered bacteria or bacteriophage to identify toxic chemical compounds, and, in some cases, to identify particular species of bacteria.
Bioreporters are genetically engineered organisms designed to detect specific compounds by incorporating a gene responsive to a selected external compound, for example by using a heterologous promoter responsive to a target compound where the promoter then induces expression of a detectable gene product in the bioreporter cell. Bioluminescent bioreporters, as used in the present context, are genetically engineered bacteria incorporating genes that when expressed result in bioluminescence. Upon detection of a specific compound, the bioreporter cell responds by producing light. A popular gene for this purpose is the lux gene. Under proper conditions, the lux genes are expressed and the subsequent bioluminescence is detectable by any of a variety of optical methods. Many of the constructs incorporated in bioluminescent bioreporter organisms derive from the bioluminescent marine bacterium Vibrio fischeri (King et al., 1990).
Sayler et al. (1998) have described bioluminescent bacterial-based bioreporters that respond to specific compounds via the production of visible light. A variety of lux-based bacterial bioreporters has been used to detect and monitor naphthalene (Heitzer et al., 1994), BTEX (benzene, toluene, ethylbenzene, and xylene) (Applegate et al., 1998), polychlorinated biphenyls (PCBs) (Layton et al., 1998), 2,4-dichlorophenoxyacetic acid (2,4-D) (Hay et al,. 2000), ammonia (Simpson et al., 2001), and the food spoilage indicator chemical xcex2-phenylethylamine (Ripp et al., 2000a).
Genetic constructs for imparting bioluminescence to bacterial bioreporter cells have generally employed a lux gene cassette derived from the marine bacterium Vibrio fischeri (Engebrecht, et al., 1983). As used herein, xe2x80x9ccassettexe2x80x9d refers to a recombinant DNA construct made from a vector and inserted DNA sequences. The complete lux cassette consists of five genes, i.e. luxA, B, C, D and E. LuxA and luxB encode the proteins that are responsible for generating bioluminescence while luxC and D encode an aldehyde required for the bioluminescence reaction.
The light response generated by bioluminescent bioreporters is typically measured with optical transducers such as photomultiplier tubes, photodiodes, microchannel plates, or charge-coupled devices. Some means of transferring the bioluminescent signal to the transducer is required, which necessitates the need for fiber optic cables, lenses or liquid light guides. Such instruments are generally unsuitable for field use. What typically results is a large, bulky instrument anchored to power and optic cables. For example, in field release experiments described by Ripp et al. (2000b), a bioluminescent bioreporter for the detection of naphthalene was used for monitoring of polyaromatic hydrocarbon degradation in soil. Bioluminescent signals were detected using a multiplexed photomultiplier tube linked to a network of fiber optic cables that proved to be expensive, fragile, and cumbersome to work with.
Battery-operated, hand-held photomultiplier units that may be interfaced with a laptop computer have been described and used in conjunction with bioreporters for field analysis of hydrocarbon contamination in groundwater (Ripp, et al., 1999a). Special bioluminescent bioreporter integrated circuits (BBICs) have been reported (Simpson, et al., 2001) and these self contained units have been shown to detect environmental contaminants such as naphthalene and BTEX by simply exposing the BBIC device to samples containing these compounds (Ripp et al, 1999b). The bioluminescent bioreporters utilized in these devices are genetically modified bacterial bioreporters that respond to specific chemicals in the environment via production of visible light.
Detection of pathogenic organisms, as opposed to chemical agents, is another area of current interest. Pathogens such as those causing human and animal diseases, foodbome pathogens and those used in biological warfare are of great significance for the safety of human populations. Furthermore, the continual appearance of new strains of bacteria underscores the need for sophisticated detection systems.
In the food industry as an example, microbial contamination of fresh fruits and vegetables has become a mounting concern during the last decade due to an increased emphasis of these products in a healthy diet and the recognition of new foodborne pathogens such as Campylobacter jejuni, Escherychia coli O157:H7, and Listeria monocytogenes (Tauxe, 1992). Federal agencies have published recommended safe food handling practices for minimizing risk; however rapid, real-time methods for detection of pathogens in the production, processing, and distribution systems are not yet available. Of particular concern in monitoring food safety is the need to identify the bacteria that cause the majority of food-related deaths in the United States, including Salmonella, Listeria monocytogenes, Escherychia coli O157:H7 and Campylobacter.
Bioluminescent methods to determine bacterial contamination are currently in use in the food industry. One technology, based on detection of ATP, relies on the biochemical requirement of all bacteria to utilize ATP for the energy production that is necessary for survival and growth. Unfortunately the ATP detection method is non-specific in nature; thus it does not differentiate among bacterial species nor does it distinguish non-pathogenic bacteria from pathogens that pose significant health risks (Vanne, et al., 1996).
Several reports have documented bioluminescent detection of a target bacterium using bacteriophage infection. Table 1 summarizes select pathogens that have been detected by these procedures.
In all of these cases, the bacteriophage contained only an incomplete lux gene, i.e. luxAB. While useful in detection of some pathogenic species, the technique suffers from several disadvantages. When only the luxAB genes are employed, an exogenous source of the aldehyde substrate for the luciferase reaction is required for detection of the bioluminescent response. This can raise problems with detection. Moreover, there are further difficulties because conditions such as the amount of added inducer may have to be adjusted. This is particularly inconvenient if the methods are used in situations such as on farms where the environment may not be conducive to running the assays and the end-user is not likely to be highly trained.
A further problem associated with bacterial detection is that often pathogens are present in very low concentrations. In such cases, existing bioluminescent methods may suffer from the disadvantage that the amount of light produced is too low to be detectable. To overcome problems of detection associated with low bacterial concentration, several non-bioluminescent detection methods are in current use. These methods often incorporate amplification procedures, such as sample pre-enrichment steps in order to elevate pathogen concentrations to detectable levels, or DNA-based polymerase chain reaction (PCR) amplification techniques. The disadvantage of these amplification steps is that they require extensive user training and expensive instrumentation.
There is therefore a need for the development of methods and devices for detecting specific bacteria, particularly pathogens, selectively, quickly, accurately and with high sensitivity. New devices are needed to provide accurate and sensitive monitoring of a variety of common pathogens, such as those implicated in health hazards associated with food and food processing, hospital environments and biological warfare.
The present invention addresses some of the deficiencies in the methods and devices presently employed in detecting individual species of bacteria, by providing a novel internally amplified bioluminescent bacteriophage/bioreporter system. In particular, the disclosed devices enable rapid and sensitive detection of specific pathogens by means of a simple-to-use fully integrated system requiring nothing more than sample addition. The sensitivity of the device is achieved by a signal amplification mechanism integrated into the design. The invention includes two cooperating elements, i.e. biosensor and bioreporter elements, that combine to operate through a novel two-step process. Biosensor elements of the invention are exemplified by genetically modified bacteriophage while the bioreporter elements may be any of a number of genetically modified cell lines. A selected pathogen, e.g., a bacterium, is infected with the biosensor bacteriophage; as a result of the infection, the bacterium produces an inducer that causes the bioreporter cell line to express the lux gene cassette, resulting in amplified bioluminescence that is readily detectable.
In particular embodiments, the invention employs bacteriophage genetically modified to carry a luxI gene. The luxI gene encodes a protein product, acyl homoserine lactone synthetase which carries out a condensation reaction of cell metabolites resulting in the production of acyl en homoserine lactone N-(3-oxohexanoyl) homoserine lactone (AHL). A selected target bacterium is infected with the genetically modified bacteriophage. Upon infection, the phage luxI gene is transcribed in the bacterium, with resultant expression of the LuxI protein by the infected target cell. AHL molecules produced in the target cell diffuse out of the target into the surrounding medium.
In the invention, infection of the target bacteria takes place in the proximity of bioreporter cells, which are genetically engineered to produce light upon stimulation by AHL. In the absence of the AHL inducer, the bioreporter cells produce little or no light. However, when the target bacteria release AHL following phage infection, AHL molecules are taken up from the surrounding medium by the bioreporter cells, and this uptake induces production of bioluminescent proteins in the bioreporter cells. This occurs because the bioreporter cell is genetically engineered to include a lux gene cassette (luxR+luxI+luxCDABE) that is responsive to AHL. AHL is an autoinducer that positively regulates the lux operon. Thus, upon stimulation with an AHL complex, the lux genes in the bioreporter cells are induced, resulting in the production of light.
A unique aspect of the invention is the amplification of bioluminescence due to the presence of the lux-modified bioreporter cells. Induction of the lux genes in one bioreporter cell results not only in the production of light-producing proteins, but also of AHL molecules. These AHL molecules then diffuse out of the light-producing bioreporter cells and further induce expression of the lux genes in neighboring bioreporter cells. This cascade effect, involving multiple neighboring bioreporter cells, results in intense bioluminescence. The infection of a target bacterium thus results in a chain reaction of bioluminescence in multiple bioreporter cells. This enables detection of very low levels of target bacteria. This novel integrative approach enables rapid pathogen detection without sample enrichment.
The bacteriophage/bioreporter system employs a luxI-integrated bacteriophage that infects only a particular bacterium. In the practice of the invention, one first selects a target bacterium, identifies a bacteriophage specific for the target bacterium and genetically engineers the bacteriophage to incorporate the luxI gene. The specificity of phage infection can be utilized to identify, detect or monitor select species of bacteria.
In most real-life situations, the target bacteria are in a natural environment, often in the presence of other microbes and various contaminants. The bacteriophage/bioreporter system addresses this problem by directing the luxI bacteriophage against specific strains of bacteria. Target bacteria may be selected from a wide variety of commonly known pathogens. Of particular interest are several types of bacteria often associated with food contamination; these include Salmonella, Escherychia coli species such as O157:H7, Listeria monocytogenes, enterobacteriaceae, as well as persistent infectious microorganisms such as Bacillus anthracis, Staphylococcus aureus and Yersinia pestis. 
Numerous other bacteria may be readily detected using the disclosed methods and devices provided that appropriate infectious phage may be identified or engineered. In practice, identification of pathogen depends on first identifying a pathogen-specific bacteriophage. Many are known; for example, bacteriophage M13 that infects E. coli. Further examples of bacteriophage that specifically infect pathogenic bacterial species are listed in Table 4.
A major consideration is the identification and genetic alteration of a species-specific bacteriophage to harbor the luxI cassette and to efficiently penetrate the bacterial cell so that the luxI can be successfully expressed in the target bacterium. A particular example is the use of bacteriophage M13 to infect E. coli. 
Certain embodiments of the invention encompass simultaneously contacting a sample with multiple bacteriophage biosensors, each of which specifically recognizes a particular bacterium and is contained within a separate sample compartment. In this way, multiple target cells of select types may be detected simultanously. The bacteriophage/bioreporter elements can be integrated onto a chip surface to provide a convenient, easily handled device.
Some embodiments of the invention encompass multi-component packaged kits containing both sensor and detector elements for detection of one or more select strains of bacteria. For sensing of select bacterial targets in a sample suspected of bacterial presence, such kits contain one or more types of genetically engineered bacteriophage, each designed to specifically infect a selected bacterium, and upon infection, to cause expression of an inducer molecule by the selected bacterium. In particular embodiments, the bacteriophage contain a luxI gene, the product of which results in formation of AHL, an inducer of the lux genes in lux-based bioluminescent cells. For detection of the infected bacterium, the kits contain a population of genetically engineered bacterial bioreporter cells capable of bioluminescence when stimulated by the inducer. In particular embodiments, the bioreporter bacteria contain a luxR-luxpro/lux I/luxCDABE gene, which is induced to produce light when stimulated by AHL. Kits may further include instructions for use, and optionally a device for measurement of the generated light, such as an integrated circuit adapted for detecting a bioluminescent signal. The integrated circuit may comprise a photodetector, low noise electronics (e.g. on-chip wireless communication system), biocompatible housing, and a semi-permeable membrane covering the bioreporter region.
The disclosed methods and devices derive from biologically-based sensor technology that can be readily adapted to pathogen detection and quality control programs. Since the required elements of the systemxe2x80x94biosensors and autoamplifying bioluminescent bioreportersxe2x80x94are completely integrated within compartments of the detector device, this system has the advantage of extreme ease of use. Operation of the system entails simply contacting a sample with a sample chamber of the device and allowing the device to process the ensuing bioluminescent signal and communicate the results. The application of this technology to important issues such as food safety and hygienic quality represents a novel method for detecting, monitoring, and preventing biological contamination.